This invention relates to mammals in which the expression of one or more genes has been suppressed, and/or one or more genes have been inserted. More specifically, the invention concerns insertion of an exogenous DNA construct into the genomic DNA of mammals thereby producing transgenic mammals with decreased or completely suppressed expression of an endogenous gene or genes, and/or with expression of a novel gene or genes.
The immune system of mammals is comprised of many specialized cells that act together in a highly complex and orchestrated manner to protect the mammal from invading pathogens, toxins, and other foreign substances.
Cells responsible for the specificity of the immune system are referred to as lymphocytes. Lymphocytes are a class of white blood cells. Two important classes of lymphocytes are T cells and B cells. T cells develop in the thymus, and are responsible for cell mediated immunity. There are many types of specialized T cells, such as for example, helper T cells (which enhance the activity of other types of white blood cells), suppressor T cells (which suppress the activity of other white blood cells), and cytotoxic T cells (which kill cells). B cells develop in the bone marrow and exert their effect by producing and secreting antibodies.
Many disorders of the immune system exist, and new disorders are continually identified. One type of commonly occurring immune disorder is the hyperactive immune system. Here, certain factors induce particular cell types in the immune system to become activated when they should not be.
Another type of immune disorder is auto immunity. This disorder is characterized by the immune system mounting an immune response against the mammal""s own tissues.
Several proteins and other molecules have been identified as having key functions in regulating the immune system response of mammals. Two such proteins are CD28 and CD45.
CD28 receptor, also known as CD28, is a homodimer of molecular weight about 44 kilodaltons (kD). CD 28 is expressed at different levels on the cell surface of various T cells, and has a molecular structure similar to receptors of the immunoglobulin supergene family. CD28 appears to be involved in regulation of T cell activation, and ultimately seems to exert this effect by regulating T cell cytokine gene expression via tyrosine phosphorylation of certain intracellular substrates such as certain phospholipases. (see Linsley et al., Ann. Rev. Immunol., 11:191-212 [1993]).
Another protein that is important in immune system regulation is the cell surface receptor molecule known as CD45 receptor or CD45. This molecule is expressed on the surface of many types of hematopoietic cells, including for example B cells and certain T cells. The gene encoding CD45 undergoes alternative splicing. CD45 has 34 exons (Johnson et al., J. Biol. Chem., 264:6220-6229 [1989]). As a result, there are multiple isoforms of CD45 (Trowbridge et al., Biochem, Biophys. Acta, 1095: 46-56 [1991], primarily due to alternative splicing of exons 4, 5, and 6. Different isoforms are expressed on different cells, but one cell type may express more than one isoform (Thomas, Ann. Rev. Immunol., 7:339-369 [1989]; Trowbridge et al., supra). CD45 has a molecular weight of between about 180kD and 235 kD, depending on the isoform. The approximately 180kD isoform, known as CD45RO, does not express exons 4, 5, or 6. CD45 is a protein tyrosine phosphatase and is involved in cell signaling (Charbonneau et al., Proc. Natl. Acad. Sci USA 85:7182-7186 [1988]; Tonks et al., Biochem., 27:8695-8701 [1988]). It has been suggested that CD45 may form a complex with proteins that are associated with antigen receptors expressed on the cell surface, and may regulate signal transduction by modulating the phosphorylation of these receptors (Justement et al., Science, 252: 1839-1842 [1991]).
Murine T cell clones lacking expression of CD45 have been generated by chemical mutagenesis (Pingel et al., Cell, 58:1055-1065 [1989]; Weaver et al., Mol. Cell. Biol., 11:4415-4422 [1991]). These cells failed to become activated (i.e., to proliferate) in response to certain compounds that normally serve as activation signals. The cells had other impaired functions as well such as decreased cytokine production.
A mutant human T cell leukemia cell line with suppressed CD45 expression has been generated using gamma irradiation (Koretzky et al., Proc. Natl.Acad. Sci. USA, 88:2037-2041 [1991]; Koretzky et al., Nature, 346: 66-68 [1990]). Among other impaired functions, this cell line was shown to lack the ability to activate a T cell receptor associated tyrosine kinase.
While the use of isolated cell lines is helpful in understanding the role of various proteins on immune system regulation, more complete information can be obtained by studying the effects of these proteins directly in a mammal (i.e., an in vivo system). To this end, various mammals have been produced that have altered levels of expression of certain genes. One class of these mammals are the so called transgenic mammals. These mammals have a novel gene or genes introduced into their genome. Another class of these mammals is the so called knockout mammals, wherein expression of an endogenous gene has been suppressed through genetic manipulation.
A variety of transgenic mammals have been developed. For example, U.S. Pat. No. 4,736,866 issued Apr. 12, 1988 describes a mouse containing a transgene encoding an oncogene.
U.S. Pat. No. 5,175,384, issued Dec. 29, 1992, describes a transgenic mouse deficient in mature T cells.
U.S. Pat. No. 5,175,383, issued Dec. 29, 1992, describes a mouse with a transgene encoding a gene in the int-2/ FGF family. This gene promotes benign prostatic hyperplasia.
U.S. Pat. No. 5,175,385, issued Dec. 29, 1992, describes a transgenic mouse with enhanced resistance to certain viruses.
PCT patent application no. WO 92/22645, published Dec. 23, 1992, describes a transgenic mouse deficient in certain lymphoid cell types.
Preparation of a knockout mammal requires first introducing a nucleic acid construct that will be used to suppress expression of a particular gene into an undifferentiated cell type termed an embryonic stem cell. This cell is then injected into a mammalian embryo, where it hopefully will be integrated into the developing embryo. The embryo is then implanted into a foster mother for the duration of gestation.
Pfeffer et al. (Cell, 73:457-467 [1993 ]) describe mice in which the gene encoding the tumor necrosis factor receptor p55 has been suppressed. The mice showed a decreased response to tumor necrosis factor signaling.
Fung-Leung et al. (Cell, 65:443-449 [1991]; J. Exp. Med., 174:1425-1429 [1991]) describe knockout mice lacking expression of the gene encoding CD8. These mice were found to have a decreased level of cytotoxic T cell response to various antigens and to certain viral pathogens such as lymphocytic choriomeningitis virus.
Kishihara et al. (Cell, 74:143-156 [1993]) describe generation of a mouse with a mutation in exon 6 of CD45. This mouse reportedly does not express CD45 in most B and T cells.
In view of the devastating effects that can result from immune disorders, there is a need in the art to provide in vivo systems for screening and evaluating drugs useful in the treatment of these disorders.
Accordingly, it is an object of this invention to provide mammals in which a gene involved in regulation of the immune system has been suppressed through the use of knockout technology, and/or a novel gene is expressed in the mammal such that the mammal may be a knockout transgenic mammal.
It is a further object of this invention to provide methods for preparing, and to prepare such knockout and knockout transgenic mammals.
These and other such objects will readily be apparent to one of ordinary skill in the art.
In one aspect, the invention provides a mouse and its progeny having a suppressed level of expression of the gene encoding CD28 on T cells. The gene may be suppressed by insertion into the genome of the mouse a nucleic acid sequence comprising at least a portion of an exon of the CD28 coding sequence linked to a marker sequence; the marker sequence can be the neomycin resistance gene.
In another aspect, the invention provides a mouse and its progeny wherein expression of the gene encoding CD45 is suppressed on B cells. The invention further provides mice with a decreased level of CD45 expression on thymocytes and peripheral T cells. Expression of the CD45 gene may be suppressed or decreased by insertion into the genome of the mouse a nucleic acid sequence comprising at least a portion of one exon of CD45 linked to a marker sequence such as the neomycin resistance gene sequence.
In a preferred embodiment, the CD45 gene that is suppressed or decreased is the CD45 exon 6 isoform.
In yet another aspect, the invention provides a mouse and its progeny wherein expression of the endogenous gene encoding CD45 isoform 6 has been suppressed, and wherein the mouse is capable of expressing a novel nucleotide sequence or transgene.
In other aspects, the invention provides embryonic stem cell lines containing a CD28 or a CD45 exon 6 isoform knockout construct.
In yet one other aspect, the invention provides a method of screening a drug for immunostimulatory effects comprising administering the drug to a mouse with a suppressed level of CD28 or CD45 expression, and assaying the mouse for immunostimulation.